Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications

Structure-Based Analysis of Five Novel Disease-Causing Mutations in 21-Hydroxylase-Deficient Patients

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90–95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients

A new locus for autosomal recessive hypercholesterolemia maps to human chromosome 15q25-q26

High serum cholesterol is an established risk factor for cardiovascular disease and is the prime target for therapeutic intervention in large groups of patients. The development of modern treatments for this major risk factor was propelled by the early realization that forms of severe hypercholesterolemia could be caused by dominantly inherited defects in the LDL receptor or in the APOB gene. Further understanding of the mechanisms contributing to early atherosclerosis will allow for new targets for therapy. We therefore identified and investigated the genetics of families from Sardinia that have recessive inheritance of precocious hypercholesterolemia. We used five families in an analysis of linkage of the autosomal recessive hypercholesterolemia locus, termed "ARH1," to chromosome 15q25-q26. A genomewide search mapped the disease-causing gene with a LOD score of 3.3 and excluded major contributions to the phenotype of other genes. A candidate gene present in the mapped chromosome region-the ligand-activated liver-transcription-factor gene ARP1 (apolipoprotein regulatory-protein gene)-has been excluded after DNA sequencing. The close-bred nature of the Sardinian population offers unique opportunities for isolation of this hypercholesterolemia-causing gene

Identification and characterization of novel PDYN mutations in dominant cerebellar ataxia cases

<p>We have recently identified missense mutations in prodynorphin (PDYN), the precursor to dynorphin opioid peptides, as the cause for spinocerebellar ataxia (SCA23) in Dutch ataxia cases. We report a screen of PDYN for mutations in 371 cerebellar ataxia cases, which had a positive family history; most are of French origin. Sequencing revealed three novel putative missense mutations and one heterozygous two-base pair deletion in four independent SCA patients. These variants were absent in 400 matched controls and are located in the highly conserved dynorphin domain. To resolve the pathogenicity of the heterozygous variants, we assessed the peptide production of the mutant PDYN proteins. Two missense mutations raised dynorphin peptide levels, the two-base pair deletion terminated dynorphin synthesis, and one missense mutation did not affect PDYN processing. Given the outcome of our functional analysis, we may have identified at least two novel PDYN mutations in a French and a Moroccan SCA patient. Our data corroborates recent work that also showed that PDYN mutations only account for a small percentage (similar to 0.1 %) of European SCA cases.</p>

μ Opioid receptor A118G polymorphism in association with striatal opioid neuropeptide gene expression in heroin abusers

μ Opioid receptors are critical for heroin dependence, and A118G SNP of the μ opioid receptor gene (OPRM1) has been linked with heroin abuse. In our population of European Caucasians (n = 118), ≈90% of 118G allelic carriers were heroin users. Postmortem brain analyses showed the OPRM1 genotype associated with transcription, translation, and processing of the human striatal opioid neuropeptide system. Whereas down-regulation of preproenkephalin and preprodynorphin genes was evident in all heroin users, the effects were exaggerated in 118G subjects and were most prominent for preproenkephalin in the nucleus accumbens shell. Reduced opioid neuropeptide transcription was accompanied by increased dynorphin and enkephalin peptide concentrations exclusively in 118G heroin subjects, suggesting that the peptide processing is associated with the OPRM1 genotype. Abnormal gene expression related to peptide convertase and ubiquitin/proteosome regulation was also evident in heroin users. Taken together, alterations in opioid neuropeptide systems might underlie enhanced opiate abuse vulnerability apparent in 118G individuals